Date of Graduation

Document Type

Program Affiliation

Degree Name

Advisor/Committee Chair

Mark Titus, PhD.

Committee Member

Robert C Bast Jr, M.D.

Committee Member

David S Hong, M.D.

Committee Member

Filip Janku, M.D., Ph.D.

Committee Member

Jennifer K Litton, M.D.

Abstract

Background

The growth and development of prostate cancer is largely driven by androgen mediated signaling. The 5-alpha reductase family of enzymes plays an essential role in the conversion of testosterone to the more potent androgen, 5-alpha dihydrotestosterone (DHT), which is capable of binding the androgen receptor to activate gene targets and downstream signaling. This study aimed to evaluate the role of 5-alpha reductase 3 (SRD5A3), a novel member of the 5-alpha reductase family, in steroid metabolism and prostate carcinogenesis.

Materials and Methods

HEK293 cells were transfected with the SRD5A3 human cDNA ORF Clone fromOriGene. Steroid substrates (testosterone, progesterone, androstenedione, epitestosterone, 11-ketotestosterone, 11α-hydroxytestosterone, 11β-hydroxytestosterone, 6β-hydroxytestosterone, and cortisol) were added to transfected HEK293 cells. The dual 5-alpha reductase 1 and 2 inhibitor, dutasteride, was added to evaluate its inhibitory activity on SRD5A3 in the presence of each steroid. Mass spectrometry analysis was used to detect and quantify the conversion of steroid substrates to their 5-alpha reduced products.

Results

SRD5A3 plasmid was successfully transfected into HEK293 cells. Mass spectrometry analysis confirmed that SRD5A3 converted testosterone to DHT. This reaction was completely inhibited by dutasteride. SRD5A3 did not convert progesterone or androstenedione to the 5-alpha reduced products. SRD5A3 had the capacity of reducing testosterone derivatives to their 5-alpha reduced products. This catalysis was concentration dependent, with higher substrate levels yielding higher amounts of the 5-alpha reduced steroid product. The inhibitory activity of dutasteride on SRD5A3 was substrate and concentration dependent.

Conclusion

SRD5A3 catalyzes the 5-alpha reduction of testosterone and testosterone analogues in the presence and absence of dutasteride. These 5-alpha reduced steroids are capable of binding and activating androgen receptor signaling. Further studies evaluating the role of SRD5A3 in prostate carcinogenesis are mandated to determine the role of therapeutic SRD5A3 inhibition across the spectrum of prostate cancer.